The invention relates to the use of derivatives of 3-amidinophenylalanine as urokinase inhibitors in particular for treating malignant tumors and the formation of metastases or as agents for targeting lymphocytes and for treating disorders of the lymphatic tissue, in particular lymphomas.
The ability of solid tumors to spread and metastasize in surrounding tissue correlates with the degradation or transformation of the extracellular matrix (tumor stroma) in the vicinity of the tumor cell and/or with the ability of said tumors to penetrate the basement membrane. Although the (patho)biochemical connections have not been completely elucidated yet, the plasminogen activator urokinase (uPA) and the urokinase receptor (uPAR) play a central role. uPA mediates the proteolytic cleavage of plasminogen to give plasmin. Plasmin in turn is a protease which has a wide range of actions and is capable of directly breaking down components of the extracellular matrix such as fibrin, fibronectin, laminin and the protein skeleton of proteoglycans. In addition, plasmin can activate xe2x80x9clatentxe2x80x9d metalloproteases and the inactive proenzyme of uPA, pro-uPA.
Tumor cells and non-malignant cells of the tumor stroma synthesize and secrete the enzymatically inactive proenzyme pro-uPA. Proteases such as, for example, plasmin or the cathepsins B and L cleave pro-uPA by limited proteolysis to give the active serine protease HMW-uPA (HMW=high molecular weight). Pro-uPA and the active protease HMW-uPA bind to the cell surface receptor uPAR (CD87). Plasmin(ogen) likewise binds to specific receptors on the plasma membranes of tumor cells which leads to focused and amplified plasminogen activation in the immediate vicinity of the tumor cells. Invasive cells thus are able to break down the extracellular matrix without finding themselves deprived of the support necessary for directed movement because of proteolysis.
Various cytobiological studies have shown that the cell-associated plasminogen activator system is of particular importance within the cascade-like reaction pathways of tumor-associated proteolytic systems (Wilhelm et al. (1994 The Urokinase/Urokinase receptor system: A new target for cancer therapy? In: Schmitt M., Graeff H., Kindermann G. (eds.): Prospects in Diagnosis and Treatment of Cancer. International Congress Series, Excerpta Medica 1050, Amsterdam, Elsevier 1994, pp 145-156). Cultures of human colon carcinoma cells showed that their ability to migrate through an extracellular matrix depended on the degree of uPA receptor saturation with active uPA. (Hollas et al., Cancer Res. 51 (1991), 3690-3695). The cell culture model likewise showed a reduction in the invasive potential of cells when PAI-1 (Cajot et al., Proc. Natl. Acad. Sci. USA 87 (1990), 6939-6943) or PAI-2 (Baker et al., Cancer Res. 50 (1990), 4676-4684) inhibited the proteolytic activity of uPA. A similar effect was achieved on inhibition of uPA binding to the cell surface by blocking the receptor by means of proteolytically inactive uPA variants (Cohen et al., Blood 78 (1991), 479-487; Kobayashi et al., Br. J. Cancer 67 (1993), 537-544). Transfection of epidermoid carcinoma cells using a plasmid expressing an antisense transcript of a part of uPAR also reduced the invasivity of said cells by suppressing uPAR synthesis (Kook, EMBO J. 13 (1994), 3983-3991). Antibodies directed against uPA and PAI-1 reduced the invasive potential of lung cancer cells in vitro (Liu et al., Int. J. Cancer 60 (1995), 501-506).
Animal models of tumors were also able to show the influence of the plasminogen activator system on the metastasizing process. Thus, addition of anti-uPA antibodies almost completely prevented the formation of lung metastases caused by human carcinoma cells in chicken embryos (Ossowski and Reich, Cell 35 (1983), 611-619). Metastasizing human carcinoma cells were transfected using an expression plasmid which encoded a proteolytically inactive, but uPAR-binding uPA mutant. The mouse model showed that carcinoma cells synthesizing inactive uPA produced a significantly smaller number of metastases after injection than nontransfected cells (Crowley et al., Proc. Natl. Acad. Sci. USA 90 (1993), 5021-5025). Moreover, after administration of uPA antisense oligonucleotides, nude mice showed inhibition of intraperitoneal spreading of human ovarian carcinoma cells (Wilhelm et al., Clin. Exp. Metast. 13 (1995), 296-302).
In recent years, the clinical relevance of factors of the plasminogen activator system (uPA, uPAR, PAI-1 and PA1-2) for the prognosis of patients having solid malignant tumors has been intensively studied. In these studies, the uPA antigen content in various tumors (e.g. breast, ovaries, stomach, lung, kidney) proved to be a strong prognostic factor both for the recurrence-free survival and for the mortality (see for example, Schmitt et al., J. Obstet. Gynaecol. 21 (1995), 151-165; Jaenicke et al., Breast Cancer Res. Treat. 24 (1993), 195-208; Kuhn et al., Gynecol. Oncol. 55 (1994), 401-409; Nekarda et al., Lancet 343 (1994), 117; Pedersen et al., Cancer Res. 54 (1994), 4671-4675). Likewise, increased concentrations of uPAR in lung cancer tissue (Pedersen et al., supra) and breast cancer tissue (Duggan et al., Int. J. Cancer 61 (1995), 597-600; Ronne et al., Breast Cancer Res. Treat. 33 (1995), 199-207) and also in the case of stomach cancer both in the tumor tissue itself (Heiss et, al., J. Clin. Oncol. 13 (1995), 2084-2093) and in tumor cells disseminated into bone marrow (Heiss et al., Nature Medicine 1 (1995), 1035-1039) correlate with a poor prognosis.
The use of synthetic uPA inhibitors makes it possible to suppress invasion and spreading of tumor cells. However, developing specific uPA inhibitors is difficult, since tissue plasminogen activator (tPA) has an identical specificity for cleaving the peptide bond Arg560/Val561 of plasminogen. In most cases therefore, low molecular weight uPA inhibitors also inhibit tPA and thus also tPA-mediated fibrinolysis. In addition, it must be guaranteed that synthetic uPA inhibitors show no strong plasmin inhibition.
Despite these restrictions, some inhibitors are known which have a certain specificity for uPA, but a low inhibition capacity, such as benzamidine derivatives and xcex2-naphthamidine derivatives, the most effective compound inhibiting uPA with Ki=2.2 xcexcmol/l (Stxc3xcrzebecher and Markwardt, Pharmazie 33 (1978), 599), or amiloride with Ki=7 xcexcmol/l (Vassalli and Belin, FEBS. Lett. 214 (1987), 187-191).
DE-A-30 35 086 discloses cyclohexanecarboxylic acid derivatives which have inhibitory effects on proteases such as trypsin, chymotrypsin, thrombin or uPA. However, the compounds studied only show quite weak and, moreover, unspecific uPA inhibition. EP-A-0 183 271 discloses lysine derivatives and the use thereof as protease inhibitors. A benzamidinolysine derivative (compound 108) is also described which inhibits uPA in vitro, but acts comparably on other proteases such as trypsin or plasma kallikrein. WO 95/17885 disloses low molecular weight polypeptides as uPA inhibitors.
Another class of known uPA inhibitors is represented by 4-substituted benzothiophene-2-carboxamidines with Ki=0.16 mmol/l in the case of benzothiophene 623 (Towle et al., Cancer Res. 53 (1993), 2553-2559). These inhibitors have a significantly higher affinity for uPA than for tPA and plasmin. uPAR-bound uPA, too, is inhibited very effectively. Disadvantageously however, the chemical synthesis of these substances is complicated and few possibilities for structural modifications are present or have been demonstrated until now.
Therefore, the development of further uPA inhibitors is very beneficial for further elucidating the role of uPA and uPAR in various diseases, especially in tumor spreading and metastasizing.
Nxcex1-Arylsulfonyl and Nxcex1-arylsulfonylaminoacyl derivatives of 3-amidinophenylalanine are known as selective inhibitors of thrombin (Markwardt et al., Thromb. Res. 17 (1980), 425-431) or of coagulation factor Xa (Stxc3xcrzebecher et al., Thromb. Res. 54 (1989), 245-252). WO 92/08709, WO 94/18185 and WO 96/05189 also disclose the use of amidinophenylalanine derivatives as inhibitors of blood clotting, in particular as inhibitors of thrombin.
Piperidides and piperazides of 3-amidinophenylalanine have been intensively studied, among which lead structures for inhibiting fibrinolytic enzymes have been found (Stxc3xcrzebecher et al., J. Enzyme Inhibition 9, 87-99, 1995; Stxc3xcrzebecher et al., J. Med. Chem. 40, 3091-3099, 1997). While Stxc3xcrzebecher et al. (1995) merely describe inhibition of thrombin, factor Xa, plasmin and trypsin, Stxc3xcrzebecher et al. (1997) also provide information about inhibiting uPA. Nxcex1-2-Naphthylsulfonyl-, Nxcex1-2-(2,2,5,7,8-pentamethylchroman-6-yl)sulfonyl- and Nxcex1-2-camphor-10-yl-sulfonyl-substituted 3-amidinophenylalaninepiperazides have a Ki for uPA of from 28 to 140 xcexcmol/l, which is about three orders of magnitude higher than the inhibition constant for thrombin. Thus it was impossible to assume that 3-amidinophenylalanine derivatives are suitable as urokinase inhibitors.
Surprisingly we have found, however, that 3-amidinophenylalanine derivatives substituted in the 2 position by a phenyl radical represent selective uPA inhibitors which are active in vivo. Furthermore, we have found that these substances have high selectivity for lymphatic tissue and thus are suitable as agents for targeting lymphocytes, for example for treating malignant disorders of the lymphatic tissue such as lymphomas.
The present invention relates to novel urokinase inhibitors of the general formula I, 
which are derived from 3-amidinophenylalanine and are present as racemates and also as L- or D-configured compounds and in which
R1 (a) is OH or OR4, where R4 is unsubstituted or substituted, for example by hydroxyl, carboxyl, sulfonyl, nitro, cyano, oxo and/or halogen, branched or unbranched C1-C8-alkyl, C3-C8-cycloalkyl or aralkyl, e.g. benzyl or phenylethyl,
(b) represents a group of the formula 
xe2x80x83in which R5 and R6 are any radicals compatible with the overall structure, where in particular
(i) R5 and R6 are H,
(ii) R5 is H and R6 is unsubstituted or substituted, for example by hydroxyl, carboxyl, sulfonyl, nitro, cyano, oxo and/or halogen, branched or unbranched C1-C8-alkyl, aralkyl, e.g. benzyl or phenylethyl, or C5-C8-cycloalkyl,
(iii) R5 and R6 are in each case independently unsubstituted or substituted, for example by hydroxyl or/and halogen, unbranched or branched C1-C4-alkyl or
(iv) R5 is H and R6 is xe2x80x94NH2 or is, in particular, an aryl-substituted or heteroaryl-substituted amino group,
(v) R5 is H or unsubstituted or substituted, for example by hydroxyl or/and halogen, unbranched or branched C1-C4-alkyl, and R6 is an amino acid residue, for example an xcex1-, xcex2- or xcfx89-amino carboxylic acid or amino sulfonic acid residue, a peptide residue, for example of up to 50 amino acids in length, or a polypeptide residue, for example of from greater than 50 amino acids to 1000 amino acids in length,
(c) represents a group of the formula 
xe2x80x83in which m is the number 1 or 2 and in which one or more of the methylene groups are optionally substituted, for example by hydroxyl, carboxyl, C1-C4-alkyl or aralkyl, e.g. benzyl or phenylethyl, with the group (c) being racemic or in D or L configuration, and R7 has the meaning of R1 in subsections (a), (b) and (f),
(d) represents a group of the formula 
xe2x80x83in which p=r=1, p=1 and r=2 or p=2 and r=1 and in which one or more of the methylene groups are optionally substituted, for example by hydroxyl, carboxyl, C1-C4-alkyl or aralkyl, e.g. benzyl or phenylethyl, and R7 has the meaning of R1 in subsections (a), (b) and (f),
(e) represents a piperidyl group which is unsubstituted or substituted in one of positions 2, 3 or 4, for example by C1-C4-alkyl, C1-C3-alkoxy or hydroxyl,
xe2x80x83where a further aromatic or cycloaliphatic ring, preferably phenyl or cyclohexyl, is optionally fused to the heterocycloaliphatic rings of the formulae (c), (d) and (e) in the 2,3 position or the 3,4 position relative to the heteroatom,
(f) represents a group of the formula 
xe2x80x83in which R8 is
(i) unsubstituted or, for example, C1-C6-alkyl-, C1-C3-alkoxy-, hydroxyl-, carboxyl, sulfonyl-, nitro-, cyano-, oxo- or/and halogen-substituted C1-C6-alkyl or aryl, such as, for example, phenyl, p-halophenyl or naphthyl,
(ii) saturated or unsaturated, branched or unbranched C1-C6-alkoxy or
(iii) unsubstituted or, for example, C1-C6-alkyl-, C1-C3-alkoxy-, hydroxyl-, carboxyl-, sulfonyl-, nitro-, cyano-, oxo- or/and halogen-substituted phenoxy or benzyloxycarbonyl,
(g) represents an acyl radical of the formula xe2x80x94COX, where X is
(i) H or unsubstituted, for example hydroxyl-, carboxyl-, sulfonyl-, nitro-, cyano-, oxo- or/and halogen-substituted, unbranched or branched alkyl, preferably C1-C6-alkyl, in particular methyl,
(ii) unsubstituted or, for example, C1-C6-alkyl-, C1-C3-alkoxy-, hydroxyl-, carboxyl-, sulfonyl-, nitro-, cyano-, oxo- or/and halogen-substituted aryl or heteroaryl, such as, for example, phenyl, p-halophenyl or thienyl, or
(iii) unsubstituted or, for example, hydroxyl-, carboxyl-, sulfonyl-, nitro-, cyano-, oxo- or/and halogen-substituted cycloalkyl, preferably C3-C10-cycloalkyl,
(h) represents aralkyl, e.g. benzyl or phenylethyl, in which the aromatic radical is unsubstituted or substituted, for example by halogen, C1-C6-alkyl, C1-C3-alkoxy, hydroxyl, cyano, carboxyl, sulfonyl or nitro,
(i) represents a carboxamide radical of the formula xe2x80x94CONRxe2x80x2Rxe2x80x3 a thiocarboxamide radical xe2x80x94CSNRxe2x80x2Rxe2x80x3, or an acetamide radical xe2x80x94CH2xe2x80x94CONRxe2x80x2Rxe2x80x3
where
(i) Rxe2x80x2 and Rxe2x80x3 are H,
(ii) Rxe2x80x2 and Rxe2x80x3 are in each case independently C1-C4-alkyl,
(iii) Rxe2x80x2 is H and Rxe2x80x3 is C1-C4-alkyl,
(iv) Rxe2x80x2 is H and Rxe2x80x3 is aryl, e.g. phenyl, or
(v) Rxe2x80x2 and Rxe2x80x3 constitute together with the nitrogen atom a heterocycloaliphatic ring having 5-7 ring members and possibly having a further heteroatom, e.g. N, O or/and S,
(j) represents SO2xe2x80x94Y where Y is
(i) unsubstituted or, for example, hydroxyl-, carboxyl-, sulfonyl-, nitro-, cyano-, oxo- or/and halogen-substituted C1-C8-alkyl, preferably methyl, trifluoromethyl, trichloromethyl,
(ii) unsubstituted or, for example, C1-C6-alkyl-, C1-C3-alkoxy-, hydroxyl-, carboxyl-, sulfonyl-, nitro-, cyano-, oxo- or/and halogen-substituted aryl or heteroaryl, such as, for example, phenyl, 4-methylphenyl, 2,4,6-trimethylphenyl, 2,4,6-triisopropylphenyl, 4-methoxy-2,3,6-trimethylphenyl, 2,2-dimethyl-6-methoxy-chromanyl, 2,2,5,7,8-pentamethylchromanyl, anthraquinonyl, naphthyl or quinolyl, or O-aryl, preferably O-phenyl or O-heteroaryl or
(iii) xe2x80x94NRxe2x80x2Rxe2x80x3, where Rxe2x80x2 and Rxe2x80x3 are in each case independently H or C1-C3-alkyl,
(k) represents a cycloaliphatic ring having from 5 to 8 carbon atoms, which is unsubstituted or substituted, for example by C1-C6-alkyl, C1-C3-alkoxy, halogen, hydroxyl or/and oxo,
(l) represents an unsubstituted or, for example, C1-C6-alkyl-, C1-C3-alkoxy-, hydroxyl-, carboxyl-, sulfonyl-, nitro-, cyano-, oxo- or/and halogen-substituted heteroaryl radical such as, for example, pyridyl or pyrimidyl, or heterocycloaliphatic radical, for example N-methylpiperidyl,
(m) represents a functionalized alkyl radical of the formula xe2x80x94(CH2)nxe2x80x94X, where the alkyl chain is unbranched or branched, n=1 to 8, and the functional radical X
(i) represents a hydroxyl group whose hydrogen atom is unsubstituted or substituted by C1-C4-alkyl, aralkyl, e.g. benzyl or phenylethyl, aryl, e.g. phenyl, C1-C4-hydroxyalkyl or acyl group CO-alkyl, (C1-C6),
(ii) is a halogen atom,
(iii) represents a tertiary amino group of the formula xe2x80x94N(alk)2, where the alkyl groups have 1 to 3 carbon atoms and are preferably the same, and the nitrogen atom may belong to a heterocyclo-aliphatic ring having 5-7 ring members and possibly having a further heteroatom, e.g. N, O or/and S,
R2 represents unsubstituted or, for example, C1-C6-alkyl-, C1-C3-alkoxy-, hydroxyl-, carboxyl-, sulfonyl-, nitro-, cyano-, oxo- or/and halogen-substituted phenyl, such as, for example, phenyl, 4-methylphenyl, 2,4,6-trimethylphenyl, 2,4,6-triisopropylphenyl, 4-methoxy-2,3,6-trimethylphenyl,
R3 is H or branched or unbranched C1-C4-alkyl, and n is 0 or 1.
The compounds may also be present as salts, preferably as physiologically acceptable acid salts, for example as salts of mineral acids, particularly preferably as hydrochlorides, or as salts of suitable organic acids.
Of the compounds defined in the general claims, those are of particular importance in which R1 corresponds to a group of the formulae (b), (d) and (f), R2 represents phenyl mono-, di- or trisubstituted by alkyl, in particular 2,4,6-substituted phenyl, e.g. 2,4,6-triisopropylphenyl, and n=0.
It is possible to prepare the compounds of the general formula I in a manner known in principle, for example as described in WO 92/08709 and WO 94/18185, and to assay their biological in vitro activity.
(L)-, (D) or (D,L)-3-cyanophenylalanine methyl ester hydrochloride is reacted with an appropriate sulfonyl chloride or a sulfonated amino acid or the halide thereof in the presence of a base to give a compound of the general formula I, which has a cyano function and in which R1=OCH3 and R2 and also R3 and n correspond to the meanings defined in the general claims. Mild acidic or alkaline hydrolysis produces therefrom the compounds of the general formula I, which have carboxylic acid structure (R1=xe2x80x94OH) and whose acid-catalyzed esterification with an appropriate alcohol leads to compounds of the general formula I, where R1=(a). Applying a method common in peptide chemistry, for example DCC in the presence of HOBt, reacting the carboxylic acids of the general formula I (R1=OH) with a nucleophile of the structures (b), (e) and (f) may give compounds with the corresponding R1 of the general formula I. To synthesize compounds with R1=(c) and (d), carboxylic acids of the general formula I with R1=OH are first reacted with cycloaliphatic amino acid esters of the structures (c) and (d), where R7 is preferably xe2x80x94OCH3 or OC2H5, the carboxylic esters obtained are hydrolyzed under mild acidic or alkaline conditions to give the corresponding carboxylic acids which may subsequently be esterified in a manner already described or be reacted with nucleophiles of the structures (b), (e) and (f), and compounds of the general formula I with R1=(c) and also (d) and with R7=(a), (b), (e) and (f) are obtained.
The target compounds of the general formula I, which have amidine structure, are obtainable from the cyano compounds in a known manner; normally, the thioamides are obtained first by addition of H2S to the cyano group, and are converted by S-methylation with methyl iodide into the thioimido esters and then into the amidino compounds by treatment with ammonium acetate in alcoholic solution. In addition and where appropriate, it is possible, using methanol or ethanol in the presence of HCl gas and, in particular cases, of an inert solvent, to prepare from the cyano compounds the corresponding imido ester hydrochlorides, which are reacted in alcoholic ammonia solution to give the amidino compounds.
The urokinase inhibitors according to the invention may be used, where appropriate, together with at least one suitable pharmaceutical excipient or carrier for producing orally, subcutaneously or intravenously administrable medicaments for controlling tumors or for diagnosis. Likewise possible is administration in combination with other active substances, for example other urokinase inhibitors such as antibodies or/and peptides.
The medicaments for controlling tumors in humans and animals may be administered topically, orally, rectally or parenterally, e.g. subcutaneously or intravenously, in the form of tablets, coated tablets, capsules, pellets, suppositories, solutions or transdermal systems such as plasters.
A particularly preferred compound of the formula (I) is Nxcex1-(2,4,6-triisopropylphenylsulfonyl)-3-amidino-(D,L)-phenylalanine 4-ethoxycarbonylpiperazide hydrochloride or the L enantiomer thereof or a pharmaceutically suitable salt of these compounds. These substances have good solubility. They are soluble in Tris buffer (pH 7.3) up to a concentration of 5xc3x9710xe2x88x925 mol/l. Addition of 5% ethanol increases the solubility to 2xc3x9710xe2x88x924 mol/l and addition of 5% DMSO to 10xe2x88x923 mol/l.
The compounds of the invention are capable of very effectively inhibiting the growth or/and spreading of malignant tumors, for example tumor spreading of pancreatic carcinoma, tumor growth of breast carcinoma and also metastasizing of tumors. It is possible to use the uPA inhibitors, where appropriate, together with other anti-tumor agents or with other types of treatment, e.g. radiation or surgery. Furthermore, the inhibitors according to the invention are also effective in other uPA-associated disorders (e.g. in preventing formation of blisters in the case of the skin disorder pemphigus vulgaris).
uPA inhibitors according to the invention are preferably characterized in that they have a Ki which is at least twofold, preferably at least 5-fold and particularly preferably at least 10-fold lower for uPA than for tPA. It is furthermore remarkable that the compounds of the invention only marginally affect blood clotting, since their Ki is too high for effective inhibition of thrombin and factor Xa.
The inventive substances of the formula I may be used in the form of conjugates with physiologically active substances, for example with radiolabels or cytotoxic agents, e.g. chemotherapeutics such as cisplatin or 5-fluorouracil, or with peptides. Furthermore it Is possible to incorporate the substances into the membrane of carrier vesicles, e.g. liposomes, and thus to facilitate targeting of active substances enclosed in the carrier vesicles, for example cytotoxic agents such as doxorubicin.
Another indication for the substances of the general formula II:
Xxe2x80x94R2,
where X is any radical, in particular an organic radical, for example a radical as defined for compounds of the formula I, but also another radical, for example a physiologically active substance such as a cytotoxic agent, a peptide or a radiolabel, a lipid or a carbohydrate, and R2 is a group as defined above, in particular 2,4,6-trisubstituted phenyl, e.g. 2,4,6-triisopropylphenyl, is the targeting of lymphocytes, which is possible owing to a 10- to 20-fold higher affinity of said substances for lymph node tissue than for other types of tissue. R2 is preferably linked to the radical X via an xe2x80x94SO2xe2x80x94 sulfonyl group. Thus, these substances are excellently suited as diagnostic agents or as agents for treating diseases of the lymphatic tissue, in particular malignant diseases such as tumor metastases and lymphomas. Administering the substances may be carried out as already described above. Diseases of the lymphatic tissue are preferably treated by administering the medicament over a number of days, for example over a period of from 5 to 20 days, followed by a treatment break and, where appropriate, by one or more administration repeats.